LOCUS       sequence-384053-        5221 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR/Cas9-mediated generic protein tagging.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5221)
  AUTHORS   Seibert M, Kurrle N, Stolp V, Nurnberger H, Tzschentke S, Borner L, 
            Wempe F, Serve H, Schnutgen F
  TITLE     Selection-free endogenous tagging of cell lines by bicistronic 
            co-expression of the surface antigen NGFR.
  JOURNAL   MethodsX. 2022 Nov 18;9:101929. doi: 10.1016/j.mex.2022.101929. 
            eCollection 2022.
  PUBMED    36444289
REFERENCE   2  (bases 1 to 5221)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "MethodsX.";
            date: "2022-11-18"; volume: "9:101929. doi"; pages: " 
            10.1016/j.mex.2022.101929. eCollection 2022"
COMMENT     Sequence Label: pGTag NGFR EGFP cLAP +0
FEATURES             Location/Qualifiers
     source          1..5221
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     7..24
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     8..24
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     34..53
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        34..52
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     263..282
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        361..436
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             549..572
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             588..608
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             624..668
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             705..728
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     primer_bind     complement(1062..1081)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             1479..1535
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     complement(2441..2461)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2441..2459)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2480..2496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2480..2496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2493..2515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2504..2520
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(2528..2534,2535..2552,2553..2558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2573..2594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2711..2728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2882..3470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2962..2981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3641..4432,4433..4501))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4264..4283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4502..4606)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4633..5088
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4720..4739)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4930..4951
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     join(5214..5221,1..15)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 cgacgttgta aaacgacggc cagtgagcgc gcgtaatacg actcactata gggcgaattg
       61 gagctacccg cattaccctg ttatccctaa gatccgacta tttcccatga ttccttcata
      121 tttgcatata cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac
      181 aaagatatta gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt
      241 tttaaaatta tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga
      301 tttcttggct ttatatatct tgtggaaagg acgaaacacc ggtatgtcgg gaacctctcc
      361 gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt
      421 ggcaccgagt cggtgctttt ttgaattcag atcttacatt tcaggacatc tagcttatcg
      481 ataccgtcga ctacgggtat gtcgggaacc tctccaggct actgaccgac gacctgccca
      541 gggcctcgga ctataaggac gacgacgaca agggcggagg cggaggggag aacctgtact
      601 ttcagggaag cggaggagga ggcaaagaaa cagctgctgc taaattcgag cggcagcaca
      661 tggacagctc tggcccctct ggcagcgccg ccaagttcag cagcctggaa gtgctgttcc
      721 agggccccct gagcagcagc ggccccagcg gcagcatggt gtccaagggc gaggaactgt
      781 tcaccggcgt ggtgcccatc ctggtggagc tggacggcga cgtgaacggc cacaagttca
      841 gcgtgtccgg cgagggcgaa ggcgacgcca cctacggcaa gctgaccctg aagttcatct
      901 gcaccaccgg caagctgccc gtgccctggc ccaccctggt gaccaccctg acctacggcg
      961 tgcagtgctt cagcagatac cccgaccaca tgaagcagca cgatttcttc aagagcgcca
     1021 tgcccgaggg ctacgtccag gagcgcacca tctttttcaa ggacgacggc aactacaaga
     1081 ccagggccga ggtgaagttc gagggcgaca cactggtgaa caggatcgag ctgaagggca
     1141 tcgacttcaa agaggacggc aacatcctgg gccacaagct ggaatacaac tacaacagcc
     1201 acaacgtgta cattatggcc gacaagcaga agaacggcat caaggtcaac ttcaagatcc
     1261 ggcacaacat cgaggacggc agcgtgcagc tggccgacca ctaccagcag aacaccccca
     1321 tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag agcgctctca
     1381 gcaaggaccc caacgagaag cgggaccaca tggtgctgaa agaatttgtg acagccgccg
     1441 gaatcaccct gggcatggac gagctgtaca agcgcggagc cacgaacttc tctctgttaa
     1501 agcaagcagg agatgttgaa gaaaaccccg ggcccatggg ggcaggtgcc accggccgcg
     1561 ccatggacgg gccgcgcctg ctgctgttgc tgcttctggg ggtgtccctt ggaggtgcca
     1621 aggaggcatg ccccacaggc ctgtacacac acagcggtga gtgctgcaaa gcctgcaacc
     1681 tgggcgaggg tgtggcccag ccttgtggag ccaaccagac cgtgtgtgag ccctgcctgg
     1741 acagcgtgac gttctccgac gtggtgagcg cgaccgagcc gtgcaagccg tgcaccgagt
     1801 gcgtggggct ccagagcatg tcggcgccgt gcgtggaggc cgacgacgcc gtgtgccgct
     1861 gcgcctacgg ctactaccag gatgagacga ctgggcgctg cgaggcgtgc cgcgtgtgcg
     1921 aggcgggctc gggcctcgtg ttctcctgcc aggacaagca gaacaccgtg tgcgaggagt
     1981 gccccgacgg cacgtattcc gacgaggcca accacgtgga cccgtgcctg ccctgcaccg
     2041 tgtgcgagga caccgagcgc cagctccgcg agtgcacacg ctgggccgac gccgagtgcg
     2101 aggagatccc tggccgttgg attacacggt ccacaccccc agagggctcg gacagcacag
     2161 cccccagcac ccaggagcct gaggcacctc cagaacaaga cctcatagcc agcacggtgg
     2221 caggtgtggt gaccacagtg atgggcagct cccagcccgt ggtgacccga ggcaccaccg
     2281 acaacctcat ccctgtctat tgctccatcc tggctgctgt ggttgtgggc cttgtggcct
     2341 acatagcctt caagaggtgg aacaggggga tcctctgacg gtatgtcggg aacctctcca
     2401 gggatgtctc gagggggggc ccggtaccca gcttttgttc cctttagtga gggttaattg
     2461 cgcgcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     2521 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     2581 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     2641 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     2701 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     2761 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     2821 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     2881 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     2941 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3001 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3061 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3121 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3181 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3241 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3301 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     3361 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3421 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3481 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     3541 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     3601 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     3661 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     3721 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     3781 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     3841 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     3901 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     3961 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4021 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4081 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4141 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4201 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4261 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4321 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4381 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4441 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4501 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     4561 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     4621 aagtgccacc tgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc
     4681 gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct ttcttccctt
     4741 cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag
     4801 ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag ggtgatggtt
     4861 cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt
     4921 tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc tcggtctatt
     4981 cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat gagctgattt
     5041 aacaaaaatt taacgcgaat tttaacaaaa tattaacgct tacaatttcc attcgccatt
     5101 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct
     5161 ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc
     5221 a
//
